Biosensors and Bioelectronics 38 (2012) 295301

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

A novel glucose biosensor using bi-enzyme incorporated

with peptide nanotubes
Byung-Wook Park a, Rui Zheng b, Kyoung-A Ko c, Brent D. Cameron b,
Do-Young Yoon d, Dong-Shik Kim a,n
a

Department of Chemical and Environmental Engineering, University of Toledo, Toledo, OH 43606, United States
Department of Bioengineering, University of Toledo, Toledo, OH 43606, United States
c
Department of Pharmacology, University of Toledo, Toledo, OH 43606, United States
d
Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea
b

a r t i c l e i n f o

abstract

Article history:
Received 29 February 2012
Received in revised form
4 June 2012
Accepted 4 June 2012
Available online 13 June 2012

A novel amperometric glucose biosensor was developed using the bio-inspired peptide nanotube (PNT)
as an encapsulation template for enzymes. Horseradish peroxidase (HRP) was encapsulated by the PNT
and glucose oxidase (GOx) was co-immobilized with the PNT on a gold nanoparticle (AuNP)-modied
electrode. A binary SAM of 3-mercaptopropionic acid (MPA) and 1-tetradecanethiol (TDT) was formed
on the surface of the electrode to immobilize the PNT and GOx. The resulting electrode appeared to
provide the enzymes with a biocompatible nanoenvironment as it sustained the enhanced enzyme
activity for an extended time and promoted possible direct electron transfer through the PNT to the
electrode. Performance of the biosensor was evaluated in terms of its detection limit, sensitivity, pH,
response time, selectivity, reproducibility, and stability in a lab setting. In addition the sensor was
tested for real samples. The composite of AuNP-SAM-PNT/HRP-GOx to fabricate a sensor electrode in
this study exhibited a linear response with glucose in the concentration range of 0.5  2.4 mM with a
R2-value of 0.994. A maximum sensitivity of 0.3 mA M  1and reproducibility (RSD) of 1.95% were
demonstrated. The PNT-encapsulated enzyme showed its retention of 4 85% of the initial current
response after one month of storage.
& 2012 Elsevier B.V. All rights reserved.

Keywords:
Glucose biosensor
Bi-enzyme
Peptide nanotube (PNT)
Binary self-assembled monolayer (SAM)
Encapsulation
Amperometric sensor

1. Introduction
The nanostructures built up of biomolecules have gained great
attraction since they show electrochemical conductivity, versatility of chemical and biological modication, and biocompatibility.
Possible fabrication of useful molecular conformation by bottomup approaches is another advantage (Adler-Abramovich et al.,
2010; Gazit, 2007; Reches and Gazit, 2006). Several biomolecules
such as peptides, lipids, DNA, RNA and proteins can be selfassembled into highly ordered nanostructures. In particular, a
peptide consisting of several to dozens of amino acids has been a
class of versatile building blocks (Yan et al., 2010). Reches and
Gazit introduced a peptide which consists of two phenylalanine
residue (PhePhe) and they showed that it forms a self-assembled
peptide nanotube (PNT). Since then, the nanostructure composed
of dipeptide residues has been regarded as a novel bio-nanostructure that can be applied to drug delivery systems, tissueengineering scaffolds, batteries, capacitors and biosensors (Beker

Corresponding author. Tel.: 1 419 530 8084; fax: 1 419 530 8086.
E-mail address: dong.kim@utoledo.edu (D.-S. Kim).

0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bios.2012.06.005

et al., 2010; Park and Kim, 2011; Ryu et al., 2010; Smith et al.,
2008; Song et al., 2004).
In this study we synthesized PNTs and use them to encapsulate the enzymes which form a sensor matrix with potential
for diabetic glucose diagnostics. When encapsulated inside a
PNT it has been shown that the enzyme activity could be
retained for an extended period of time even under unfavorable
conditions for enzymes (Yu et al., 2005). The unique properties
of PNT, such as excellent biocompatibility and electrical conductivity, provide enzymes with advantages of stable immobilization, protection from harsh environments, and superior
signal transfer. Recently, it has been demonstrated that peptide
nanostructures in biosensors can facilitate the detection of
various compounds including hydrogen peroxide (Yemini
et al., 2005a), NADH (Yuan et al., 2011), ethanol (Yemini
et al., 2005b), phenolic compounds (Kim et al., 2011a), organophosphates (Kim et al., 2011b), Pb (Rica. et al., 2010) and Cu
(Viguier et al., 2011).
For glucose detection, in general, glucose oxidase (GOx) is used
in glucose sensors. The detection mechanism is based on the
electrochemical oxidation of hydrogen peroxide produced from
the oxidation of glucose at a high potential with help of GOx

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(Fu et al., 2011). However, at a high potential, various oxidative

compounds, such as uric acid and ascorbic acid that are commonly present in biological samples may produce undesirable
interfering reactions for the quantication of the glucose
(Benedetto et al., 1996). One of the approaches to overcome this
problem is to use a bi-enzyme system of GOx and horseradish
peroxidase (HRP) (Ghindilis and Kurochkin, 1994). A combination
of GOx and HRP has been mostly applied to design a biosensor
system for glucose detection (Delvaux et al., 2005; Li et al., 2009;
Manesh et al., 2010).
To enhance the enzyme activity and stability, one of the
enzymes was encapsulated by PNTs. Encapsulating HRP by a
PNT was proven to be successfully in our previous study (Park
et al., 2010a, b). Also, the activity of encapsulated HRP was
observed to be further retained in comparison with free enzymes
in a solution (Park et al., 2012). Motivated by these observations,
the combination of the PNT-encapsulated HRP and GOx was
examined as the main part of an amperometric biosensor for
diabetes. To the best of our knowledge, there has been no report
on a glucose sensor using binary enzymes, one of which is
encapsulated into the PNT. In addition, the gold nanoparticle
(AuNP) modied electrode and a binary SAMs were used in this
system to immobilize the PNT and GOx on the electrode. It was
supposed to enhance the biosensor performance and the attachment of the PNT onto the surface of the gold electrode (Park et al.,
2011a, b; Zheng et al., 2011). The measurement was performed in
the presence of an electron mediator hydroquinone. The optimized conditions for analytical performance of the developed
biosensor were studied. The stability and reproducibility of the
sensor were also evaluated. The results obtained in this study
indicated the validity of the biosensor incorporated with the PNT
encapsulation.

2. Material and methods

2.1. Materials and reagents
HRP (E.C 1.11.1.7, 59 Umg-1, from Horseradish), GOx (E.C
1.1.3.4, 192 Umg-1, from Aspergillus niger), D-()-glucose,
3-mercaptopropionic acid (MPA), 1-tetradecanethiol (TDT),
Gold(III) chloride trihydrate (HAuCl4), N-hydroxysuccinimide
(NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrodhloride (EDC) and Naon (5 wt%) were purchased from Aldrich
and Fluka (Sigma, USA) and used without further purication.
Potassium hydroxide (KOH), potassium nitrate (KNO3), potassium
ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6),
potassium chloride (KCl), hydrogen peroxide (H2O2), sulfuric acid
(H2SO4), sodium phosphate monobasic (H2NaO4P) and sodium
phosphate dibasic (HNa2O4P) were analytical grade from Fisher
Scientic (Fisher, USA) and used without further purication. All
solutions were prepared with water puried to 418 MW with a
Barnstead Nanopure system (Barnstead Co., USA) ltered through
a 200 mm lter paper.
2.2. Apparatus and measurements
Cyclic voltammetry (CV) and differential pulse voltammetry
(DPV) were performed using a Gamry Reference 600 potentiostat
(Gamry Instruments, USA) with Gamry PHE 200 and PV 220
physical electrochemistry software programs. A three-electrode
system in a conventional glass-made cell was used for electrochemical measurements with a bare polycrystalline gold
electrode of 3 mm diameter as a working electrode, a platinum
wire as an auxiliary electrode, and a Ag9AgCl9KCl (saturated)
electrode as a reference electrode. All the potential values

reported are given with respect to this reference electrode. All

measurements were made in a Gamry VistaShield Faraday cage
(Gamry Instruments, USA).
Electrochemical impedance spectroscopy (EIS) measurements
were carried out using a Gamry Reference 600 potentiostat in
5 mM Fe(CN)36  /Fe(CN)46  solution with 0.1 M KCl as a supporting
electrolyte. Impedance spectra were collected in the frequency
range from 0.1 Hz to 100 kHz with a potential amplitude of
5 mV rms at 10 points per decade. EIS results were analyzed by
tting the experimental impedance data to electrical equivalent
circuit models. Parameters of the electrical equivalent circuits
were obtained by tting the impedance function to the measured
Bode and Nyquist plots with a complex nonlinear least square
(CNLS) program built in the Gamry EIS 300 electrochemical
impedance spectroscope.
The instrument used for characterization of the AuNP deposited on the bare gold electrode and the PNT modied electrode
was eld emission scanning electron microscopy (FE-SEM) (Hitachi S-4800, Japan). The electrodes were examined at 2 kV acceleration voltages.

2.3. Electrode preparation

2.3.1. Encapsulation of the enzyme into a PNT
The PNT was prepared by dissolving a lyophilized form of the
dipeptide of 1,1,1,3,3,3-hexauoro-2-propanol at 100 mg ml  1.
The self-assembly of the PNT was carried out at the optimal
concentration of 2 mg ml  1. It was known that the concentration
is a crucial factor for forming self-assembled PNTs (Reches and
Gazit, 2003). To encapsulate HRP inside the PNT, rst 1 ml of the
PNT solution in a vial was placed in an oven at 35 1C to dry
completely. Next, 1 ml of 50 mM phosphate buffer solution (PB,
pH 7.0) containing HRP at the concentration of 1 mg ml  1 was
added into the vial that contained the dried PNT. It was then
mixed using a vortex mixer subsequently. Finally, the enzymePNT solution was incubated in a shaker at 5 1C, 120 rpm for a
week. It is thought that the HRP was encapsulated inside the
hollow structure of the PNT due to the capillary effect (Yu et al.,
2005; Yang et al. 2004) and stabilized on the inner surface of PNT
(Park et al., 2010b).
In order to obtain the solution containing the HRP encapsulated into the PNT, the micro centrifugation was carried out
at 16,000 g for 10 min. After separating the PNT from the
solution, the PNT was thoroughly washed three times with the
PB solution to remove the excess HRP (Park et al., 2010b).

2.3.2. Gold nanoparticle modied electrode

Gold electrodes were mechanically polished with 0.5, 0.3, and
0.05 mm sizes of alumina suspensions on a polishing microcloth
(Buehler, USA). Then they were sonicated in DI water and ethanol
for 5 min. Polycrystalline gold electrodes were treated by piranha
solution (a mixture with 3:1 of concentrated sulfuric acid and 30%
hydrogen peroxide) for 15 min. Piranha solution is extremely
reactive with organic materials and has to be used with sufcient
protection. The pretreated clean gold electrodes were immersed
in 0.1 M KNO3 solution containing 3 mM HAuCl4. Electrochemical
deposition of gold nanoparticles (AuNP) on a planar gold
electrode was carried out at the working potential of 200 mV
vs. Ag9AgCl (satd KCl) for 330 s (Park et al., 2011a). Previously, it
was conrmed that an increase in the surface area of the
electrode could be performed by the deposition of 3D structural
AuNP (Park et al., 2011b).

B.-W. Park et al. / Biosensors and Bioelectronics 38 (2012) 295301

2.3.3. Formation of a binary self-assembled monolayer onto the

AuNP electrode
Thiol solution was prepared by mixing 1 mM ethanol solutions
of MPA and TDT at a ratio of 9:1 while keeping the total
concentration of the binary SAM at 1 mM (Park et al., 2011a, b).
The binary SAM of MPA and TDT was formed on the AuNP electrode
by soaking the electrodes into the mixed thiol solution for 1 h. The
electrodes were subsequently rinsed in ethanol and DI water.
2.3.4. Fabrication of AuNP-SAMs-PNT/HRP-GOx electrode
AuNP-SAMs modied electrode was immersed in a solution of
1 mM EDC and 3 mM NHS for 1 h to activate the terminal
carboxylic acid groups of MPA. In this step, EDC converts the
carboxyl group into a reactive intermediate, which is susceptible
to attack by amines of the PNT. After the activation and rinsing the
electrodes with the PB solution, a 10 ml of the PNT/HRP solution
was immediately placed onto the electrode surface and then was
allowed to dry at 4 1C. Then, a 10 ml of 3 mg ml  1 GOx solution
spread evenly onto the AuNP-SAMs-PNT/HRP electrode and was
allowed to dry at 4 1C. Finally a 1 ml of 0.5 wt% Naon solution was
dropped on top of the AuNP-SAMs-PNT/HRP-GOx electrode. After
drying, the electrode was immersed in the PB solution to wash
away the non-immobilized residuals. All the modied electrodes
were stored at 4 1C when not in use. The procedure for preparing
glucose sensor is schematically shown in Fig. 1.
2.4. Real sample measurements
The sensor prepared in this work examined with human serum
samples (Atlanta biologicals, USA). Each sample was diluted 10
times in the buffer solution. The diluted samples were then
analyzed by the developed biosensor at a working potential of
0.03 V. The measured current was converted to the glucose
concentration by using the calibration curve. The results were
compared with those obtained YSI 2300 STAT plus glucose
analyzer (YSI life sciences, USA).
The optimum response of the electrode in amperometric measurements was determined by measuring the current responses of
0.5 mM glucose in 50 mM PBS at pH values, 5.8, 6.4, 7, 7.7, 8.1.

297

3. Results and discussion

3.1. Characterization of the AuNP-SAMs-PNT/HRP-GOx electrode
The surface morphology of the gold electrodes was observed
by FE-SEM. Fig. 1 shows the representative schematic for the
modied electrodes and the SEM images of each layer. Fig. 1A and
B show nanoscale images of the bare Au electrode and AuNPSAMs modied electrode surfaces. It was found that gold nanoparticles were successfully formed on the surface of electrode,
showing a relatively rough surface. Fig. 1C represents that the
PNT/HRP was immobilized onto the AuNP-SAMs modied electrode surface. It is thought that the immobilization mechanism
could be taken place not only through amine coupling (EDC/NHS
chemistry) but also hydrophobic interaction between the PNT and
the terminal methyl group of TDT.
3.2. Electrochemical behavior of the modied electrode
We used electrochemical impedance spectroscopy (EIS), cyclic
voltammetry (CV) and differential pulse voltammetry (DPV) to
characterize the electrochemical behavior of the modied
electrode.
3.2.1. Electrochemical impedance spectroscopy
EIS is an effective and non-destructive method for characterizing the interfacial electrical properties of modied surfaces of
electrodes. This method can provide the information such as
resistance and capacitance of an electrode.
Fig. 2 compares the Nyquist plots of the modied electrodes in
Fe(CN)36  /Fe(CN)46  . The equivalent circuit (inset of Fig. 2A) is
chosen to t the impedance data. The AuNP modied gold
electrode exhibits an almost straight line (Fig. 2A (curve a)) that
is a very low Ret of 90 O and that is characteristics of the diffusion
limited electron-transfer process. After the binary SAM of MPA
and TDT is deposited, the larger semicircle (Fig. 2A (curve b)) with
an interfacial resistance of 759 O, due to the formation of the
SAM layer on the electrode surface. Moreover, TDT, consisted of
CH3 and SH, resulted in the hydrophobic surface area. Upon

Fig. 1. The representative schematic for the modied electrode and the SEM images of each layer. SEM images of (A) Bare Au, (B) AuNP-SAMs, (C) AuNP-SAMs-PNT and
(D) AuNP-SAMs-PNT-Nf. Scale bar 500 nm (A), (B) and (C) and 300 mm (D).

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Fig. 2. Comparison of the Nyquist plots of the electrodes and the electron transfer resistance of the each layer. (A) Nyquist plots of impedance spectra obtained in the
presence of 5 mM Fe(CN)36  /Fe(CN)46  with 50 mM PB solution (pH 7.0) and 0.1 M KCl at (a) AuNP, (b) AuNP-SAMs and (c) AuNP-SAMs-PNT/HRP-GOx electrode. The
frequency range from 0.1 Hz to 100 kHz with a potential amplitude of 5 mV rms at 10 points per decade. The inset shows the equivalent circuit applied to t the data.
(B) The values of electron transfer resistance (Ret) of the each layer.

Fig. 3. Effects of glucose on the electrochemical response. (A) Cyclic voltammogram of AuNP-SAMs-PNT/HRP-GOx electrode (a) in the absence and (b) in the presence of
0.5 mM glucose. (B) Differential pulse voltammograms of the electrode (a) in the absence and (b) in the presence of 0.5 mM glucose. (C) Cyclic voltammograms of the
electrode at different scan rates (25200 mV s  1). (D) Anodic and cathodic current plotted against the scan rate.

immobilization of PNT/HRP and GOx over the AuNP-SAMs modied electrode, the Ret for the redox process of the probe
drastically increased to 4464 O (Fig. 2A (curve c)). This is due to
the attachment of the PNT and enzymes that increases the extent
of insulation of the conductive support by the hydrophobic
materials.

the DPV was carried out, an obvious difference could be observed

(Fig. 3B).
The enzymatic reaction mechanism of the AuNP-SAMs-PNT/
HRP-GOx electrode developed here is described as follows:
HRP (Red) H2O2-HRP (Ox) 2H2O
HRP(Ox)HQ-HRP (Red)BQ

3.2.2. Voltammetry
Upon the addition of glucose, the AuNP-SAMs-PNT/HRP-GOx
electrode triggers bioelectrocatalytic reaction, which changes the
cyclic voltammogram with the cathodic current (Fig. 3A). The
increase in the current is caused by the cascade of reactions
taking place at the electrode surface. The DPV experiments have
also been performed, which corresponded to the CV results. When

BQ2e  2H -HQ
The substrate, glucose reacts with GOx, in the presence of the
natural co-substrate O2, to generate hydrogen peroxide (H2O2).
The H2O2 serves as substrate for HRP, resulting in the oxidized
state of HRP which can be reduced by the redox mediator
hydroquinone (HQ).

B.-W. Park et al. / Biosensors and Bioelectronics 38 (2012) 295301

Moreover, the peak currents of the AuNP-SAMs-PNT/HRP-GOx

electrode are shown to be dependent on the scan rate (Fig. 3C).
The anodic and cathodic peak currents increase linearly with the
scan rate, with correlation coefcients (R2) values of 0.9893 and
0.9949 for the anodic and cathodic peaks, respectively, in the scan
rate range of 25200 mV s  1. This linearity between peak current
and scan rate shows that the redox reactions are surface-conned
electrode reactions.
3.3. Effect of pH and operational potential effects on response of the
bi-enzyme electrode
The pH of the buffer solution is essential to the sensitivity of
biosensors. The pH affects the enzyme activity and the electrochemical behavior of GOx. It was known that the range of the
optimal pH for GOx is 6.57.5, which can vary with immobilization
methods. The experimental results in the current study showed
that the response signal increases with pH up until pH 7, and then it
decreases at a higher pH range (3071.5 nA at pH 5.8, 4572 nA at
pH 6.4, 7872.3 nA at pH 7, 6372 nA at pH 7.7 and 4171.3 nA at
pH 8.1). The low current response at the high and low pH ranges
could possibly be raised by decreasing the enzyme activity. It
turned out that the optimum pH for the biosensor developed would
be pH 7. For further studies the PB solution with pH 7 was used as a
working electrolyte for glucose detection.
3.4. Amperometric response to glucose
Amperometric measurements are widely used to evaluate the
performance of enzyme-based sensors. Amperometric responses of
the electrode were collected at a constantly applied potential of
0.03 V vs. Ag9AgCl electrode. Upon the each injection of 0.2 mM
glucose in a range of 0.28.0 mM at regular intervals, the response

299

shows a rapid and prominent increase in current, as shown in

Fig. 4A. The biosensor response reaches 95% of a steady-state value
within about 910 s at each glucose injection. The response time
shows that there is no serious diffusion barrier through the hollow
structure of the PNT. This response time is also comparable to that of
10 s for biosensor based on SBA-15 mesopores (Dai et al., 2008) and
that of 8 s for carbon nanotube (CNT) biosensors (Zhu et al., 2007).
The corresponding calibration curve of the glucose sensor
exhibits a linear response in the concentration range from 0.5 to
2.4 mM with a correlation coefcient of 0.994, which is compatible with that of 0.032.43 mM (Zhu et al., 2007). The detection
limit was estimated to be 73.1 mM based on 3s (where s is the
standard deviation of the blank solution, number of experiments10), which is better than the reported value of 100 mM
(Manesh et al., 2010). In the linear range, the slope (i.e., sensitivity) of 0.3 mA M  1 was determined, which is higher than that of
the reported value of 0.17 mA M  1 (Benedetto et al., 1996;
Manesh et al., 2010).
Fig. 4B also shows that the saturation of response current can
be observed for the electrode in the calibration curve, which is
consistent with the characteristic of the MichaelisMenten
kinetics. The apparent MichaelisMenten constant (Km), which
is a reection of the enzymatic afnity and an essential parameter
to describe the enzyme-substrate kinetics of the biosensor, can be
calculated from the electrochemical version of the Lineweaver
Burk plot (Fig. 4C) according to the following equation:
 
1
1
K app 1

m
Iss
Imax Imax c
where Iss is the steady-state current after the addition of the
substrate, c is the bulk concentration of the substrate and Imax is
the maximum current measured under saturated substrate condition. The K app
m value was determined by the graphical analysis of
the slope and intercept for the plot of the reciprocals of the steady

Fig. 4. Amperometric resonse of the electrode and its kinectics analyses. (A) Amperometric response of the AuNP-SAMs-PNT/HRP-GOx electrode upon successive addition
of 0.2 mM glucose into 50 mM PB solution (pH 7.0) at 0.03 V. (B) Calibration curve of glucose sensor. (C) LineweaverBurk plot.

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B.-W. Park et al. / Biosensors and Bioelectronics 38 (2012) 295301

state current versus glucose concentration. The value of the K app

m
was calculated 11 mM which suggested that the apparent afnity
between the immobilized GOx and substrate be higher. This
behavior of the sensor turns out to be governed by ideal
MichaelisMenten enzyme kinetics, which suggests that the
enzymatic reaction rate constitute the rate-limiting step in the
biosensor response. The observed K app
value for the developed
m
biosensor is lower than that in other previously reported bienzyme glucose sensor (49 mM) based on the use of poly(toluidine blue O) lm. In this report, toluidine blue O was applied as a
mediator and polymerized on multi-walled carbon nanotube
(MWNT) modied glassy carbon electrode to improve the performance of the biosensor (Wang et al., 2010). The lower value
implies the strong afnity of substrate to the enzymes, resulting
in a facilitated enzymatic reaction. It may be noted that the K app
m
value depends on the nature of the immobilization matrix and the
immobilization method.
3.5. Selectivity and interferences
The real sample for glucose detection contains other sugars
and common species that could potentially interfere with the
glucose detection. The interferences selected for the test are
ascorbic acid (AS), sucrose (SU), galactose (GA), and lactose (LA).
In light of low concentrations of these sugars in the blood, the
interference needs to be seriously considered because their
molecular structures are similar to that of glucose. Furthermore,
because ascorbic acid could be easily oxidized at a relatively high
overpotential, a lower working potential helps avoid the electrochemical reaction of the interferences. As shown in Fig. 5A., the
current responses to 0.5 mM of AS, SU, GA and LA are negligible
compared to 0.2 and 0.5 mM of glucose. The results are due to the

excellent selectivity of the AuNP-SAMs-PNT/HRP-GOx electrode

and the lower working potential. Moreover, the Naon acting as
an effective permselective layer could eliminate the interferences
and increase the selectivity of the developed biosensor.
3.6. Reproducibility and stability
The precision of the developed bio-sensing system was characterized in terms of repeatability and reproducibility. For the
repeatability, successive amperometric measurements with the
same electrode were carried out. Sets of eight successive calibrations for 0.5 mM glucose were constructed yielding a relative
standard deviation (RSD) of 1.95% for their slopes. Furthermore,
the reproducibility of the sensor measurements was investigated.
The RSD for three different electrodes using the same conditions
was 5.74% corresponding to 0.5 mM glucose. These results show
that the fabrication procedure of the biosensor is reliable and that
the modied electrodes are reproducible.
Long-term stability is a basic requirement for the development
of biosensors. The stability of the AuNP-SAMs-PNT/HRP-GOx
electrode was evaluated and its response to 0.5 mM of glucose
was measured for one month. The electrode, when not in use, was
stored in the PB solution at 4 1C. The results in Fig. 5.B showed
that 85% of the initial response remained after a storage of one
month. The decrease in the current response may be due to a loss
of the enzyme activity in long-time keeping. However, the
excellent performance of the sensor after one month storage
could be attributed to the following aspects: Good long-term
stability of this sensor system can be attributed to the excellent
biocompatibility of the PNT for retaining the enzyme activity.
Also, it is possibly that the hollow structure of the PNT provides
mild environment around the enzymes. Table 1 shows the

Fig. 5. Stability analyses of the electrode. (A) Inuence of electroactive interferences of ascorbic acid (AS, 0.5 mM), sucrose (SU, 0.5 mM), galactose (GA, 0.5 mM) and
lactose (LA, 0.5 mM). The working potential 0.03 V. (B) Long-term stability of the AuNP-SAMs-PNT/HRP-GOx electrode stored in the PB solution (pH 7.0) at 41C.
Table 1
Comparison of the performance of various bienzymatic glucose biosensors.
Components of biosensor

Sensitivity
(mA M  1)

Stability
(days/percent
of the initial response)

References

AuNP-SAMs-PNT/HRP-GOx
ITO/PTMSPA/HRP-GOx
GC/CNT/HRP-GOx/PTBO
Au/CS/Con A/HRP-GOx
GC/SBA-15/HRP-GOx
Au/SWNT/HRP-GOx/PPy
PC/Au/MPE/GA/HRP/GA/GOx
GC/HRP-GOx/PPy
GE/CB/HRP-GOx

0.3
0.16
113
1.18
90
1.03
400
0.17

30/85
50/55
25/75
35/89
60/60
14/67

4/75
40/50

Current work
Manesh et al. (2010)
Wang et al. (2010)
Li et al. (2009)
Dai et al. (2008)
Zhu et al. (2007)
Delvaux et al. (2005)
Benedetto et al. (1996)
Ghindilis and Kurochkin (1994)

B.-W. Park et al. / Biosensors and Bioelectronics 38 (2012) 295301

Table 2
Determination of glucose concentration contained in human serum samples.
Sample
no.

Determined by
biosensora (mM)

Given by glucose
analyzerb (mM)

Relative
deviation (%)

1
2
3

0.546 7 0.051
0.368 7 0.012
0.618 7 0.069

0.504
0.370
0.594

7.69
0.54
3.88

a
b

Mean7 S.D. of three measurements.

Value obtained by the commercial glucose analyzer.

characteristics of AuNP-SAMs-PNT/HRP-GOx in terms of sensitivity and stability are compared with earlier reported bi-enzymatic
glucose biosensors.
3.7. Real sample analysis
The practical usage of the developed glucose biosensor was
carried out with human serum samples. A series of human serum
samples were analyzed. The sample was 10-fold diluted with
50 mM PB at pH 7. The diluted samples were then analyzed by the
developed biosensor at a working potential of 0.03 V. The measured current was converted to the glucose concentration by
using the calibration curve. The results were compared with those
obtained YSI 2300 STAT plus glucose analyzer (YSI life sciences,
USA). In Table 2, the values obtained from our biosensor system
are close to those from the analyzer. It can be concluded that the
values from the glucose sensor developed are in good agreement
with those from the commercial instrument for the glucose
detection, demonstrating that the developed sensor has a great
potential for the practical usage for real clinical samples.

4. Conclusions
The results demonstrate the possible use of the PNT to conveniently fabricate a new bi-enzyme glucose biosensor. The encapsulation of HRP inside the PNT was shown to provide a spatially
biocompatible environment on the electrode surface, which could
further retain the enzyme activity for an extended period of time.
Moreover, the spatially aligned aromatic compounds were considered to enhance the electron transfer. Therefore, the developed
glucose sensor appears to exhibit reasonable sensitivity, reproducibility and long-term stability. The methodology applied in this study
possibly provides a new platform for the fabrication of biocompatible glucose sensor and other types of biosensors.

Acknowledgments
The CMSC (Center for materials and sensor characterization) at
University of Toledo is acknowledged for the use of instruments.

301

Financial support from Nano-STAR Center at Kwangwoon University, and Kwangwoon University (2011) is gratefully acknowledged.

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